Over the past several decades, the incidence of melanoma has increased at a faster rate than that of any other solid tumor (1). The highest have been observed in Australia and New Zealand (27.9/100,000 among males and 25.0 among females) and in North America (10.9/100,000 among males and 7.7 among females). In 2001, it was estimated that 51 400 cases of invasive melanoma would be diagnosed (2) Early recognition and surgical excision of the primary tumor provide the best opportunity for obtaining a cure. However, prognosis associated with more advanced melanoma remains poor. Patients presenting with thick primary lesions, American Joint Committee on Cancer (AJCC) melanoma stage IIB/C, and those with regional nodal metastases (AJCC melanoma stage III) have a reported 5-year survival ranging from 30 to 70%. This is related to the high failure rates associated with surgical therapy alone in locally and regionally advanced cases. The risk of recurrence after surgery has been reported to be as high as 60% for patients with melanoma stage IIB/C and 75% for patients with melanoma stage III (3). Compounding this, it has been the lack of effective adjuvant therapy, particularly the limited efficacy of cytotoxic chemotherapeutic agents, against melanoma. Recently, the use of high-dose interferon in the adjuvant setting has been reported to improve both disease-free and overall survival (4,5). The benefits of interferon, however, are still being debated, and treatment with interferon is not without significant cost, risk and toxicity. The results achieved with interferon highlight the potential for the immune system to prevent recurrence after surgical resection of high-risk melanomas.
Melanoma has emerged as the primary model for developing immunotherapies for several reasons. Histopathologic evidence of tumor regression is frequently observed within primary melanoma specimens, along with the presence of tumor infiltrating lymphocytes, thus suggesting a prominent role for the immune system in melanoma (6). Melanoma cells readily adapt to tissue culture, resulting in the creation of panels of melanoma cell lines to study. The paucity of effective therapies (chemotherapy, radiation) has resulted in a lower threshold for testing immunological therapies in patients with melanoma (7,8).
For all these reasons, there has been a significant effort to treat malignant melanoma using immunologic modalities. The use of immunotherapy can be categorized as either active or passive. Passive immunotherapy is the use of either antibodies or cells that have previously been sensitized to host tumor antigens. The host need not mount an immune response, the agent will directly or indirectly mediate tumor killing. Active immunotherapy on the other hand is the use of agents that will cause the host to mount an immune response. This can further be broken down to nonspecific and specific active immunotherapies. Nonspecific agents are those that stimulate the immune system globally, but do not recruit specific effector cells. Specific active immunotherapy is designed to elicit an immune response to one or more tumor antigens.
Current strategies for the immunotherapy of melanoma include the induction or enhancement of immune responses against tumor antigens presented by melanosomal proteins such as tyrosinase, tyrosinase related proteins TRP1 and TRP2, gp100, and MART-1. Potent immune responses in humans and animals against melanocytes and these antigens, comprising melanocyte eradicating CTL responses as well as humoral responses, have been observed in autoimmune depigmenting disorders.
Vitiligo is one such acquired depigmenting disorder, and is characterized by the loss of melanocytes from the epidermis. Several types of vitiligo are distinguished according to the distribution of the achromic lesions. One or more lesions in a quasidermatomal pattern are characteristic for unilateral vitiligo while this unilateral distribution is absent in focal vitiligo. Both are localized types of vitiligo. Generalized vitiligo is characterized by multiple scattered lesions in a symmetrical distribution pattern. The course of the disease is unpredictable but is often progressive with phases of stabilized depigmentation (9). An extending vitiligo with enlarging lesions or development of new lesions is defined as active vitiligo.
The association with autoimmune disorders and organ specific antibodies as well as the fact that non-surgical repigmenting therapies have immune-modulating effects also support the idea of an autoimmune pathogenesis of the disease. The humoral antibodies are generally considered to be an epiphenomenon. Progress in the understanding of the pathogenesis of vitiligo emerges from studies on the local phenomena leading to or related with the process of depigmentation. The normal appearing skin adjacent to the depigmented area is histologically characterized by degenerative changes in melanocytes, vacuolar changes of basal cells, the presence of a lymphocytic infiltration in epidermis and dermis as well as melanophages in the upper dermis (10-12). In progressing inflammatory vitiligo, which is characterized by achromic lesions surrounded by a red raised rim, the lymphocytic infiltration proceeds in the direction of skin that still contains melanocytes, suggesting a role of the inflammatory infiltrate in melanocyte disappearance (13). A recent study localized CLA+ cytotoxic T cells in apposition to disappearing melanocytes in the perilesional skin of generalized vitiligo. Also, a focal, epidermal expression of ICAM-1 and HLA-DR at the interaction site between skin homing T cells and melanocytes was detected (14). HLA-DR expression implicates the involvement of MHC class II-restricted T cells in the pathogenic process (15). Perilesional T-cell clones (TCC) derived from patients with vitiligo exhibited a predominant Type-1-like cytokine secretion profile, whereas the degree of Type-1 polarization in uninvolved skin-derived TCC correlated with the process of microscopically observed melanocyte destruction in situ. Detailed analysis of broad spectrum of cytokines produced by perilesional- and nonlesional-derived CD4+ and CD8+ TCC confirmed polarization toward Type-1-like in both CD4 and CD8 compartments, which paralleled depigmentation process observed locally in the skin. Furthermore, CD8+ TCC derived from two patients also were analyzed for reactivity against autologous melanocytes. The antimelanocyte cytotoxic reactivity was observed among CD8+ TCC isolated from perilesional biopsies of two patients with vitiligo. Finally, in two of five patients, tetramer analysis revealed presence of high frequencies of Mart-1-specific CD8 T cells in T-cell lines derived from perilesional skin (16).
One distinctive form of vitiligo is contact or occupational vitiligo (17,18). This form is unique in that its onset correlates with exposure to certain chemicals that induce chemical leukoderma. Contact/occupational vitiligo is distinct from chemical leukoderma in that the initial cutaneous depigmentation extends from the site of chemical contact and subsequently develops into progressive, generalized vitiligo (19). There is anecdotal and experimental evidence demonstrating that certain environmental chemicals are selectively toxic to melanocytes, both in culture and in vivo (20,21,22) and are thus responsible for instigating vitiligo (19). The majority of these toxins are aromatic or aliphatic derivatives of monophenols and benzenediols, containing a phenylring substituted with 1 or 2 hydroxyl moieties, which may be in the ortho-(1 and 2, catechols), meta-(1 and 3; 1,3 benzenediol is also referred to as resorcinols) and para-(1 and 4) configurations, such as para-hydroxybenzene, also referred to as hydroquinone (FIG. 1). Table 1 lists a selection of preferred monophenols, benzenediols and/or catechols (or 1,2 dihydroxyphenyl compounds) and sulfhydryls capable of depigmenting skin and/or instigating vitiligo. Some of these compounds have been added to bleaching creams, products used to remove hyperpigmented lesions. Interestingly, these creams are not toxic to melanocytes from all individuals. Even at high dosages only a subset of humans depigment in response to application. Exposure of the skin to certain phenols and catechols such as Monobenzyl ether of hydroquinone (MBEH), 4-tert-butylphenol (TBP) and 4-tert-butylcatechol (TBC) causes leukoderma and can induce vitiligo-like depigmentation. Many of the cases have been reported by workers who were exposed to these compounds in the polymer or leather industries. MBEH, TBP, TBC and other monophenols or benzenediols are substrate analogs that can be oxidized by enzymes having tyrosinase activity, yielding quinones and in particular orthoquinone intermediates, compounds that are highly reactive and which rapidly react with cystein and/or histidine moieties in proteins. In particular, some high reactive orthoquinones will immediately react with cysteine or preferably histidine residues moieties in the vicinity of or more preferably within the catalytic site of the tyrosinase enzyme.
Thus far the results of treatment for metastatic melanoma have been disappointing. Single-agent chemotherapy produces response rates ranging from 8% to 15%, and combination chemotherapy, from 10% to 30%. These responses are usually not durable. Immunotherapy, using interferon (IFNγ) or particularly high-dose interleukin (IL)-2, has also shown a low response rate of approximately 15%, although it is often longer-lasting. In fact, a small but finite cure rate of about 5% has been reported with high-dose IL-2. Phase II studies of the combination of cisplatin-based chemotherapy with IL-2 and interferon-alfa, referred to as biochemotherapy, have shown overall response rates ranging from 40% to 60%, with durable complete remissions in approximately 8% to 10% of patients. Although the results of the phase II single-institution studies were encouraging, phase III multicenter studies have reported conflicting results, which overall have been predominantly negative. Moreover, IL-2 and IFN administration are associated with multiple side effects, and only physicians experienced in the management of such therapies should administer them.
Riley (23,24) applied the depigmenting phenol compound 4-HA (4-hydroxyanisole) as a chemotherapeutic in melanoma, without success. Attempts to use these agents for the treatment of disseminated melanoma have foundered on problems due to unfavorable pharmacokinetics, primary toxicity or pharmacological actions of analogue substrates, and toxicity of hepatic metabolites. The intra arterial infusions in the lower limbs gave rise to serious renal and hepatic toxicity.
Novel strategies are clearly needed to improve the clinical outcome of melanoma. The use of the autoantigens responsible for the autoimmune disorder vitiligo for the induction of an anti-tumor response has since long been investigated. So far, this has not yielded improved therapies and medicaments for the treatment of melanoma. Similarly, the studies of Riley and others concerning the use of compounds capable of inducing occupational vitiligo and cytotoxicity against melanocytes for the treatment of melanoma have not been successful. The current inventors aimed to overcome the current status quo. The current invention is based on new insights in how antigens present in melanocytes may be chemically modified and activated in situ, providing new methods and means for the treatment of tyrosinase expressing malignancies such as melanoma.